The present invention provides novel polynucleotides and polypeptides encoded by such polynucleotides. This invention relates generally to the field of learning-induced protein kinases.
Classical conditioning of the eyeblink/nictitating membrane (NM) response is a useful behavioral model for investigating the neural substrates underlying basic associative learning and memory (Thompson and Krupa, 1994). Several lines of evidence have pointed to the cerebellum as a critical structure in the formation and storage of the memory trace in eyeblink conditioning. The neural output from the cerebellar (interpositus) deep nuclei to target structures forms the essential efferent pathway in this type of learning (Thompson, 1986; Krupa et al., 1993; Kim et al., 1997). The cerebellum receives projections from mossy fibers and climbing fibers, which are thought to convey information about the conditioned stimulus (CS) and unconditioned stimulus (US), respectively. The CS and US signals converge in the cerebellar cortex and deep nuclei (Ito, 1984; Thompson, 1986). The convergence of these two inputs also fulfills the condition required for the induction of cerebellar long-term depression (LTD) (Ito, 1989; Linden and Conner, 1995).
Recent studies of eyeblink conditioning in various gene knockout mice have shown that deficits in cerebellar LTD correlated with impairment in eyeblink conditioning (Aiba et al., 1994; Shibuki et al., 1996). Purkinje cell degeneration (pcd) mutant mice, which lack cortical efferents to the deep nuclei, exhibited a marked impairment in eyeblink conditioning (Chen et al., 1996), but significant learning did occur. Substantial impairment in eyeblink conditioning has also been reported in rabbits with lesions limited to cerebellar cortex (Lavond et al., 1987; Lavond and Steinmetz, 1989a; Yeo et al., 1985b). In contrast, lesions of the cerebellar deep nuclei, in particular the dorsal anterior interpositus nucleus (IN), completely prevented learning in naive animals and permanently abolished conditioned responses (CRs) in well-trained animals (Krupa, 1993; McCormick et al., 1982; Lincoln et al., 1982; Yeo et al., 1985a; Clark et al., 1992; Steinmetz et al., 1992). Such effective lesions had no persisting effects on any aspect of the reflex unconditioned response (UR) (Steinmetz et al., 1992; Ivkovich et al., 1993). Finally, reversible inactivation of the IN during training completely prevented learning but reversible inactivation of the immediate output from the IN, the superior cerebellar peduncle, and its target, the red nucleus, did not prevent learning at all (Clark and Lavond, 1996; Clark et al., 1992; Krupa et al., 1993; Krupa and Thompson, 1995; 1997; Nordholm et al., 1993); Thus, within the neural circuitry involved in eyeblink conditioning, the IN represents a critical locus for the acquisition and expression of the CR.
Much experimental evidence also supports the idea that the synthesis of new RNAs or proteins are necessary for long-term changes in synaptic efficacy associated with long-term memory formation (Agranoff, 1967; Barondes and Cohen, 1966; Davis and Squire, 1984). For example, transcription and translation inhibitors blocked long term facilitation of the gill-withdrawal reflex in Aplysia (Montarolo et al., 1986). Long term memory was further shown to require the activation of the transcription factor CREB in some animal models (Kaang et al., 1993; Bourtchuladze et al., 1994; Yin et al., 1994). As for eyeblink conditioning, infusioh of the protein synthesis inhibitor anisomycin into the IN interfered with conditioning in the rabbit (Bracha and Bloedel, 1996). In this patent, we report our studies to further unravel the molecular pathways underlying eyeblink conditioning. We first demonstrated that inhibition of RNA synthesis in the interpositus nuclear cells interfered with the acquisition of CRs. Next, we applied the method of differential display PCR (DD-PCR) (Liang and Pardee, 1992) to examine changes in gene expression that accompanied eyeblink conditioning. We identified RNA molecules that were induced with conditioning. The cDNA cloning and sequence analyses showed that the expressed gene was the KKIAMRE kinase, a member of the cdc2-related and MAP kinase family.
The invention describes the cDNA sequence, the deduced amino acid sequence, and the genomic sequence of a learning-induced kinase, KKIAMRE kinase, expressed in rabbit brain during classical conditioning.
Polynucleotides include those with sequences substantially equivalent to including fragments thereof. Polynucleotides of the present invention also include, but are not limited to, polynucleotides complementary to the aforementioned cDNA and genomic DNA polynucleotide sequences.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers for PCR, use as reagents to identify homologous genomic sequences, mRNA sequences, or cDNA sequences in the same or different species, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of antisense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue MRNA using, e.g., in situ hybridization.
The polypeptides according to the invention can be used in a variety of procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention cane be used to generate an antibody that specifically binds the polypeptide. The polypeptides of the invention also can be used for the study of mechanisms of learning and memory.
Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention or a polynucleotide of the present invention and a pharmaceutically acceptable carrier. In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, as part of methods for improving learning and memory in normal subjects or to ameliorate learning and memory deficits in impaired subjects. In one embodiment, a polynucleotide of the invention is delivered to a patient in an appropriate vector that directs expression of the polypeptide in neuronal tissue.
The methods of the present invention further relate to the methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample. Such methods can, for example, be utilized as a prognostic indicator of learning and memory impairment syndromes or deficits in affected subjects.